1. Technical Field of the Invention
The present invention relates to novel skin equivalents, to a method for the production thereof and to the epidermis equivalents and dermis equivalents comprising such novel skin equivalents (themselves per se novel).
2. Description of the Prior Art
For several years, the perfection of models of reconstructed skin which permit conducting studies required for a better understanding of the role of the skin in the mechanical domain and physiological domain has been ongoing.
Thus, models which more or less approximate human skin have been developed. Exemplary are the models described in EP-A-285471, EP-A-285474, EP-A-789074, EP-A-502172, EP-A-418035, WO-A-9116010, EP-A-197090, EP-A-20753, FR-A-2665175 and FR-A-2689904.
Very generally, the models of reconstructed skin described in the aforesaid publications comprise human keratinocytes associated or otherwise with other skin cells such as melanocytes and/or Langerhans cells, deposited onto a support, often a dermis equivalent, and cultured under conditions such that they commence a program of differentiation which results in the formation of an epidermis equivalent.
The dermis equivalents described to date are either artificial membranes such as, for example, Millipore filters, collagen-based subcutaneous substitutes, plastic or any other support which is compatible with cell viability, or supports which are more developed such as to approximate natural dermis, for example previously de-epidermilized dermis or collagen/fibroblast mixed lattices.
In the collagen/fibroblast mixed lattices the association of native collagen and isolated human fibroblasts ultimately provides a dermis equivalent which mimics a dermis which has not been subjected to the actions of the weather.
The protocols employed for the preparation of said lattices use collagen originating in general from young tissues, this collagen having, however, been subjected to the important post-translational modifications which intervene in the complex but nevertheless normal processes of its biosynthesis, but not having undergone all of the modifications which can intervene, in particular, during aging.
It is known to this art, in particular, to this art, that during aging and during the progress of certain diseases such as diabetes, non-enzymatic processes operate which involve an ose (glucose or ribose) which reacts according to the Maillard reaction with an amino group (for example a lysine residue) of the collagen to form a Schiff's base. This base, after undergoing an Amadori molecular rearrangement, may provide, by a succession of reactions, intramolecular bridging such as, for example, of pentosidine type. This phenomenon, termed glycation of collagen, increases uniformly with age, leading to a uniform increase in the glycation-product content of the skin. These glycation products include, for example, pyrraline, carboxymethyl lysine, pentosidine, crosslines, Nε-(2 carboxyethyl) lysine (CEL), glyoxal-lysine dimer (GOLD), methylglyoxal-lysine dimer (MOLD), 3DG-ARG imidazolone, versperlysines A, B, C, threosidine or, alternatively, advanced glycosylation end products or AGEs. This phenomenon is amplified in certain disease states, such as, for example, diabetes.
Without wishing to be bound to any particular theory as regards aging of the skin, it should be noted that other characteristics which might also be a consequence of these glycation phenomena, such as a decrease in heat denaturation, an increase in resistance to enzymatic digestion and an increase in intermolecular bridging, have been demonstrated during aging of the skin (Tanaka S. et al., 1988, J. Mol. Biol., 203, 495-505; Takahashi M. et al., 1995, Analytical Biochemistry, 232, 158-162). In addition, modifications due to the glycation of certain constituents of the basal membrane such as collagen IV, laminine and fibronectin have been demonstrated (Tarsio J F. et al., 1985, Diabetes, 34, 477-484; Tarsio J F. et al., 1988, Diabetes, 37, 532-539; Sternberg M. et al., 1995, C.R. Soc. Biol., 189, 967-985).
Thus, it is understood that during aging of the skin the physicochemical properties of collagen are modified and such collagen becomes more difficult to dissolve and more difficult to degrade.
Thus, one of the components of aged skin indeed appears to be glycated collagen.
It is also very well known that the skin constitutes a close association between at least two components thereof, i.e., the epidermis and the dermis. The interactions between the dermis and the eipdermis are such that it is reasonable to consider that a modification of one may have consequences on the other. It may be suspected that the aging of the dermis, in particular with its glycation phenomena, can only have consequences for the epidermis to which it is associated. Thus, during skin aging, the glycation of the collagen must promote modifications of the epidermis which must participate in the aging of the epidermis.
In this respect, it has now been demonstrated that a constitutive protein of normal epidermis, i.e., the β1 type integrin, an extracellular matrix receptor (see Ruoslahti E., 1991, Cell Biology of Extracellular Matrix, Plenum press New York, 343363), shows a distribution of its expression in aged epidermis which is very different from that in young epidermis. Specifically, while in a young epidermis, i.e., for the purposes of this invention an epidermis from a young subject, this protein is expressed in the very deep layers of the epidermis, i.e., up to a maximum of the second suprabasal layer, the situation is completely different in an aged epidermis, i.e., for the purposes of this invention in an epidermis from an elderly subject, where this protein is expressed in most layers of the epidermis, directly through to the last suprabasal layers under the cornified layer.
To date, no model of reconstructed skin in vitro is capable, either because of the protocols of preparation technique, or the simple fact that once reconstituted it does not undergo modification, of producing a skin equivalent at least one of the constituents of which provides one of the components of skin aging. Thus, no model of reconstructed skin in vitro presents the properties of an aged skin or permits the study of the processes resulting therein, or the study of the compounds and/or compositions which would at least make it possible to slow down or retard this/these process(es). The only known evaluations of these phenomena entail in vivo studies, either in animals or humans. Most particularly, for ethical reasons, the advantage of having such a model is conspicuously apparent.
Studies on glycation are known in the prior art. For example, a method is described for obtaining a connective tissue equivalent in the form of a lattice of glycated collagen and fibroblasts (see in this respect Frey et al. (1992, C.R. Soc. Biol., 187, 223-231). However, not only have Frey et al. never investigated or even suggested the possibility of preparing a skin equivalent from their lattice, they have never compared this lattice to any dermis equivalent. In addition, but while recognizing the validity of the conjunctive tissue model of Frey et al., it will be appreciated that the protocol employed cannot provide the objectives established hereby, i.e., to reproduce in vitro a skin, and consequently an epidermis and a dermis, which has all or part of the properties of an aged skin.
By incubating the collagen and sugar for 9 hours at a temperature of 4° C., Frey et al. initiate the collagen glycation reaction, this reaction then continuing in the lattice in which the collagen is in this manner preglycated. If it is desired to attain a sufficient level of glycation, such as to mimic an aged skin, it is then necessary to permit the glycation reaction to continue in the lattice formed in this fashion, i.e., in the process of contraction, for a further time which is sufficient to attain the desired level. However, those skilled in this art are cognizant that to establish an epidermis equivalent containing at least keratinocytes on a dermis equivalent of collagen/fibroblast lattice type, the seeding of the keratinocytes must be conducted onto a lattice which cannot have exceeded a determined stage of contraction. It will thus be seen, therefore, that it is not possible to obtain by the Frey protocol a dermis equivalent which mimics an aged or very aged dermis, i.e., highly or very highly glycated.